Ultrasound-mediated vascular gene transfection by cavitation of endothelial-targeted cationic microbubbles

Abstract

Objectives: Ultrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection.

Background: Contrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound.

Methods: Cationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)-stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging.

Results: Charge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm(2)). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1-targeted microbubbles and by ultrasound molecular imaging of P-selectin-targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin-targeted microbubbles but was associated with vascular rupture and hemorrhage. At 0.6 MPa, there were no adverse bioeffects, and transfection was 5-fold greater with P-selectin-targeted microbubbles.

Conclusions: We conclude that ultrasound-mediated transfection at safe acoustic pressures can be markedly augmented by endothelial juxtaposition.

Figure 1. Properties of Lipid Microbubble Carriers

(A) Fluorescent microscopy (excitation filter 530 to 560 nm) of SYBR-gold–labeled plasmid cDNA charge coupled to cationic microbubbles (scale bar = 5 μm). (B) Mean ± SD amount of cDNA charge coupled to the surface of different microbubble preparations. Biot = presence of DSPE-PEG-biotin. *p < 0.01 versus both neutral preparations. (C) Example of an acoustic amplitude response caused by microbubble cavitation during ultrasound exposure at 1 MPa using 30-cycle ultrasound pulses every 20 ms (50 Hz). A portion of the pressure waveform for the second pulse is expanded. (D) Mean ± SD amplitude of microbubble response (peak-to-peak) measured by a passive cavitation detector for cationic microbubbles with and without cDNA during sequential 1-MHz ultrasound pulses at 0.5- and 1.0-MPa peak negative acoustic pressure. The eventual stabilization after approximately pulse 10 was not significantly different from that obtained with saline alone (Online Fig. 1).

Figure 2. Microbubble Adhesion Efficiency

(A) In vitro microbubble attachment density per optical field (OF) to streptavidin-coated plates in a flow chamber. Data are displayed for shear stresses of 0.6 and 1.5 dyne/cm². *p < 0.05 versus both the corresponding neutral biotinylated preparation and the cationic biotinylated preparation with cDNA. (B) Illustrative image and quantitative data for in vivo attachment of cationic nontargeted or ICAM-1–targeted microbubbles observed by intravital microscopy in TNF-α–treated cremasteric vessels. The image illustrates retention of DiI-labeled ICAM-1–targeted microbubbles in a venule running vertically across the image (scale bar: 20 μm); *p < 0.05 versus corresponding non-targeted agent.

Figure 3. Molecular Imaging of Microbubble Attachment Efficiency

(A) Quantitative acoustic signal intensity (AU) corrected for freely circulating contrast for nontargeted control. *p < 0.05 versus corresponding nontargeted agent. (B) Examples of contrast-enhanced ultrasound images obtained of the post-ischemic hindlimb skeletal muscle 8 min after intravenous injection for control (Co) or P-selectin–targeted (Targ) microbubbles. Images are color coded (scale at left) and subtracted for freely circulating agent.

Figure 4. Targeted Transfection of Luciferase Reporter Plasmid

(A) In vivo optical imaging data of luciferase reporter gene transfection 3 days after CUMGD, quantified as photon flux at the site of ultrasound exposure 10 min after intraperitoneal injection of luciferin. PNAP = peak negative acoustic pressure. Transfection was significantly lower at 0.6 MPa compared with 1.0 and 1.8 MPa for both agents. (B) Examples illustrating luminescence (color-coded) 3 days after bilateral hindlimb ischemia and intravenous injection of either nontargeted or P-selectin–targeted cDNA-coupled cationic microbubbles during ultrasound at 0.6 MPa. Ventral surface depiliation was performed to reduce light attenuation. (C) Immunohistochemistry for luciferase with peroxidase illustrating transfection (arrows) of venular and capillary endothelium and perivascular cells. Scale bar = 50 μm.

Figure 5. Bioeffects From CUMGD Measured Immediately After Ultrasound Exposure

(A) Examples showing no petechiae or moderate petechiae at the site of ultrasound exposure at peak negative acoustic pressures (PNAP) of 0.6 and 1.8 MPa, respectively, and percentage of animals with each petechia score. (B) Quantitative fluorescence for Texas Red–dextran from the ultrasound-exposed and control leg immediately after contrast ultrasound-mediated gene delivery (CUMGD). Examples of in vivo optical imaging (color-coded and superimposed on bright light images) are shown at the right with a mid-line shield placed to eliminate bladder signal. (C) Masson’s trichrome imaging illustrating absence of significant fibrosis (blue) other than normal perivascular pattern in tissue exposed to microbubbles and ultrasound at 0.6 or 1.8 MPa.